Mitochondrial Performance Enhancement Nanoemulsion Method

ABSTRACT

An aqueous, intra-oral, nanoemulsion blend is provided that enhances mitochondrial performance in mammals when orally administered. The blend includes at least two different monolayer surfactant bound particle components and at least one bilayer water-core liposome component. The blend optionally may include a micelle.

REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. Nonprovisional application Ser.No. 16/214,289, filed Dec. 10, 2018, entitled “Mitochondrial PerformanceEnhancement Nanoemulsion,” which claims the benefit of U.S. ProvisionalApplication No. 62/596,338 entitled “Compositions 85 Methods forEnhancing Mitochondrial Performance” filed Dec. 8, 2017, both of whichare incorporated by reference in the entirety.

BACKGROUND

Negative health and disease states are associated with low mitochondrialcellular content and mitochondria damage in the cells of mammals. Suchnegative health and disease states include cardiomyopathy, lacticacidosis, developmental delay, failure to thrive, impaired neurologicalfunction, and obesity. The obesity link is believed attributable to manyof the genes that encode for mitochondrial proteins being inverselycorrelated with body mass.

Pyrroloquinoline quinone disodium salt (PQQ), resveratrol, genistein,hydroxytyrosol, and quercetin are reported to stimulate mitochondrialbiogenesis (the formation of new mitochondria) and to improvemitochondrial respiratory control. Resveratrol and genistein arebelieved to affect cell-signaling pathways important for mitochondrialbiogenesis, but lack the water solubility of PQQ. PQQ is believed toprotect brain, other nerve, and heart tissue against damage from oxygenradicals and is known to increase growth in animal cells, bacteria, andplants.

PQQ is naturally found in kiwifruit and human breast milk at pico- tonano-molar concentrations. As an ingested solid, it is proposed thatmilligram quantities of PQQ are needed per kilogram of diet to provideenhanced mitochondrial biogenesis. Micromolar concentrations of PQQ perkilogram of diet are believed necessary to provide enhancedmitochondrial biogenesis when PQQ is injected. Unlike Vitamin C, PQQ canundergo thousands of redox cycles without degradation or polymerization.PQQ is also somewhat unique in that it can be detected in tissue withlittle or no dietary exposure.

Prior studies have demonstrated that mice and rats fed diets lacking PQQhave reduced mitochondrial content. Conversely, Hepa1-6 mouse cellsexposed to 10-30 μM PQQ for 24-48 hours demonstrated increased citratesynthase and cytochrome c oxidase activity, Mitotracker staining,mitochondrial DNA content, and cellular oxygen respiration. PQQ also wasshown to protect cells from mitochondrial inhibition by rotenone,3-nitropropionic acid, antimycin A, and sodium azide. J. Biol. Chem.,Jan. 1, 2010; 285(1): 142-152.

In animals, dietary PQQ deprivation results in abnormal development,immune dysfunction, and decreased reproductive performance. While PQQ isbelieved to affect many genes, the most affected genes are believedthose involved in mitochondrial-related function. For example, in amouse example, PQQ deficient mice have a 20 to 30% reduction in therelative amount of mitochondria in the liver when compared to PQQsupplemented mice.

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is produced by severalplants in response to injury or, when the plant is under attack bypathogens, such as bacteria or fungi. Sources of resveratrol in foodinclude the skin of grapes, blueberries, raspberries, and mulberries.Resveratrol exists as two geometric isomers: cis-(Z) and trans-(E). Thetrans- and cis-resveratrol isomers can be either free or bound toglucose. While 70% of orally administered resveratrol is absorbed by thebody, the bioavailability is only about 0.5% as resveratrol isextensively metabolized into its glucuronide conjugate by the liver andintestine before reaching the bloodstream. In animal models, it isbelieved that from 200 to 500 milligrams (mg) of resveratrol is neededper kilogram of diet to enhance mitochondrial biogenesis.

Coenzyme Q10 (CoQ10) is present in animals and most bacteria. CoQ10 isconsidered to have structural similarity to a vitamin and is present inall respiring eukaryotic cells, primarily in the mitochondria. It is acomponent of the mitochondrial electron transport chain and participatesin aerobic cellular respiration, which generates energy in the form ofATP. Therefore, those organs with the highest energy requirements—suchas the heart, liver, and kidney—have the highest CoQ10 concentrations asthey have the highest concentration of mitochondria. Having three redoxstates, CoQ10 can act as a two or one electron carrier in themitochondrial electron transport chain. Thus, CoQ10 can provide a singleelectron, as needed for electron transport to iron-sulfur clusters, andcan also function to scavenge (deactivate) free radicals, such as oxygenradicals. While readily measured in blood plasma, this is an indicationof dietary CoQ10 intake, not tissue concentration. The body can produceCoQ10 similarly to cholesterol, but CoQ10 synthesis may be inhibited bysome beta-blockers, blood-pressure reduction medication, and severelylimited by statins. When consumed orally, CoQ10 has an approximate 10%bioavailability.

Vitamin E includes the tocotrienols (alpha, beta, gamma, and deltaisomers) and the tocopherols (alpha, beta, gamma, and delta isomers),which are all oil-soluble. All tocotrienol and tocopherol isomers (thus,all forms of Vitamin E) have some antioxidant activity due to an abilityto donate a hydrogen atom (a proton plus electron) to an oxygenradical—thus deactivating the radical by forming an —OH (alcohol) group.The critical chemical structural difference between the tocotrienol andtocopherol forms of Vitamin E is that the tocopherols have saturatedside chains, while the tocotrienols have unsaturated isoprenoid sidechains with three double bonds. The different tocotrienol isomersdemonstrate different bioavailability and efficacy depending on the typeof antioxidant performance being measured. Conventionally,alpha-tocopherol has been the preferred form of “Vitamin E”, as thisoil-soluble tocopherol isomer is credited with having the highestantioxidant biological activity and is preferentially absorbed andaccumulated in humans when orally consumed.

Adaptogens or adaptogenic substances describe compounds that whenadministered to a living organism decrease cellular sensitivity tostress. While adaptogens may have multiple pathways of action, a primaryrole of an adaptogen is to reduce the cortisol response, thus the body'sproduction of cortisol in response to stress. Humans produce cortisol inresponse to stress and/or low blood sugar. Cortisol will increase bloodsugar through increased metabolism of fat, protein, and carbohydrate,while simultaneously suppressing the immune system and bone growth.

The effect of cortisol on the immune system is substantial, as evidencedby the hydrocortisone form of cortisol being used as a medication toreduce unwanted immune response, such as skin inflammation and rash.Elevated levels of cortisol arising from the stress response have knowndeleterious effects on the immune system. For example, wounds from punchbiopsies of students took an average of 40% longer to heal whenperformed three days before an examination as opposed to biopsiesperformed on the same students during summer vacation.

FIG. 1A and FIG. 1B represent a liposome 100 having a double wall(bilayer) of phospholipids formed from a hydrophilic exterior wall 120and a hydrophilic interior wall 125. The interior of the double wall 110is hydrophobic. The hydrophilic interior wall 125 forms a capsuleinterior 130, to form what may be referred to as a “water-core”liposome. Liposomes may be thought of as small, fluid-filled capsuleswhere the wall of the capsule is formed from two layers of aphospholipid. As phospholipids make up the outer membranes of livingcells, the liposome 100 can be thought of as having an outer, permeablemembrane wall like a cell, but without a nucleus or the other componentsof a living cell within the capsule interior 130. The outer and innerwalls 120, 125 of the represented liposome 100 are water-soluble, whilethe interior of the wall 125 is fat-soluble. A common phospholipid usedto form liposomes is phosphatidylcholine (PC), a material found inlecithin.

When introduced to the body, liposomes are known to deliver theirinternal contents to living cells through one of four methods:adsorption, endocytosis, lipid exchange, and fusion. In adsorption, theouter wall of the liposome sticks to the living cell and releases itscontents through the outer wall of the living cell into the living cell.In endocytosis, the living cell consumes the liposome, thus bringing theentire liposome into the cell. The cell then dissolves the outer wall ofthe liposome and releases the liposome contents into the interior of theliving cell. In lipid exchange, the liposome opens in close proximity tothe living cell and the living cell takes in the localized highconcentration of liposome interior. In fusion, the outer wall of theliposome becomes part of the outer wall of the living cell, thuscarrying the contents of the liposome into the enlarged living cell.These pathways allow for a potential 100% transfer of the interiorcontents of the liposome to the interior of the living cell, if theliposome can be brought into close proximity to the cell and is properlyconstructed to interact with the target cell.

FIG. 2 represents a flattened side view of the double wall (bilayer) ofphospholipids that forms the liposome. The phospholipids have polar,hydrophilic “heads” and less polar, relatively hydrophobic “tails”. Inthis representation, the heads form the top and bottom of the bilayer,with the tails forming the interior middle. Oil-soluble compounds canreside between the top and bottom layers within the interior areaoccupied by the tails.

FIG. 3 represents a micelle 300 having a single wall of phospholipids(monolayer) forming a hydrophilic exterior 320 and a hydrophobicinterior 310 lacking the hydrophilic capsule interior of a liposome.Thus, in relation to a liposome, a micelle lacks a bilayer and does notprovide the capsule interior that can contain a water-soluble,hydrophilic core composition. The micelle 300 may be thought of as theouter wall of a liposome without the inner wall providing for a capsuleinterior. Polyethylene glycol modified vitamin E, such as tocopherylpolyethylene glycol succinate 1000 (TPGS), may be used to form micellesin water as the TPGS has a water-soluble head and an oil-soluble tail.

FIG. 4 represents a monolayer surfactant where the oil component isassociated with the hydrophobic tails of a surfactant. In thisrepresentation, the surfactant has formed a circular shape, thusencircling the oil component and approximating a relatively large,expanded micelle, but that is not required for the oil component toassociate with the hydrophobic tails.

Nutritional supplements are conventionally introduced to the bloodstreamin multiple ways. Supplements taken orally are adsorbed at differentrates due to many factors. For example, on average about 10% to 20% of asolid supplement taken orally is adsorbed. This can be increased toabout 30% with an orally taken gel capsule, to about 45% with atransdermal patch, and to about 50% with a conventional intra-oral(sublingual). Injections provide from approximately 90% to 100%adsorption, but are uncommonly used for nutritional supplements.

The present invention avoids or ameliorates at least some of thedisadvantages of conventional oral supplement preparations intended toenhance mitochondrial performance in a living organism.

SUMMARY

In one aspect, the invention provides an aqueous, intra-oral,nanoemulsion blend for enhancing mitochondrial performance in animalswhen orally administered, the blend includes at least two differentmonolayer surfactant bound particle components, where a first particlecomponent includes at least one amphiphilic fat, a polyethylene glycolsurfactant form, an associating oil, and a cell-signaling pathwayenhancement composition, and a second particle component includes atleast one amphiphilic fat, a polyethylene glycol surfactant form, anassociating oil, and coenzyme Q10; and at least one bilayer water-coreliposome particle component, where the at least one bilayer water-coreliposome particle component includes at least one amphiphilic fatforming a capsule interior, where the capsule interior includes apyrroloquinoline quinone salt dissolved in water

In another aspect of the invention, there is an aqueous, intra-oral,nanoemulsion blend for enhancing mitochondrial performance in animalswhen orally administered, the blend including at least two differentmonolayer surfactant bound particle components, where a first particlecomponent comprises at least one amphiphilic fat, a polyethylene glycolsurfactant form, an associating oil, and a cell-signaling pathwayenhancement composition, and a second particle component comprises atleast one amphiphilic fat, a polyethylene glycol surfactant form, anassociating oil, and coenzyme Q10; and at least one bilayer water-coreliposome component, where the at least one bilayer water-core liposomecomponent includes at least one amphiphilic fat forming a capsuleinterior, where the capsule interior includes an adaptogenic herbextract in water, the adaptogenic herb extract including water- andoil-soluble adaptogenic herbs.

In another aspect of the invention, there is an aqueous, intra-oral,nanoemulsion blend for enhancing mitochondrial performance in an animalwhen orally administered, the blend including a cell-signaling pathwayenhancement composition delivery means for delivering a cell-signalingpathway enhancement composition to the bloodstream of the animal; acoenzyme Q10 delivery means for delivering coenzyme Q10 to thebloodstream of the animal; and a pyrroloquinoline quinone salt deliverymeans for delivering a pyrroloquinoline quinone salt to the bloodstreamof the animal, where when administered intra-orally to an animal thecell-signaling pathway enhancement composition delivery means, thecoenzyme Q10 delivery means, and the pyrroloquinoline quinone saltdelivery means in combination are configured to provide a totalbloodstream delivered concentration ratio for the pyrroloquinolinequinone salt to the cell-signaling pathway enhancement composition from1:1 to 1:2 and for the pyrroloquinoline quinone disodium salt to thecoenzyme Q10 from 1:2 to 1:4.

In another aspect of the invention, there is a method of making anaqueous, intra-oral, nanoemulsion blend for enhancing mitochondrialperformance in animals when orally administered, the method includeshomogenizing a mixture comprising a cell-signaling pathway enhancementcomposition, a first portion of amphiphilic fat including at least 30%by weight phosphatidylcholine, a polyethylene glycol surfactant form, anassociating oil, glycerin, ethanol, and water to form a first emulsion;homogenizing a mixture comprising coenzyme Q10, a second portion ofamphiphilic fat including at least 30% by weight phosphatidylcholine, apolyethylene glycol surfactant form, an associating oil, glycerin,ethanol, and water to form a second emulsion; combining the first andsecond emulsions with at least one tocotrienol isomer of Vitamin E toform a third emulsion; combining a third portion of amphiphilic fatincluding at least 30% by weight phosphatidylcholine with glycerin andethanol in water to form micellular amphiphilic fat; combining anadaptogenic herb extract including oil- and water-soluble herbs, water,ethanol, and glycerin with a fourth portion of amphiphilic fat includingat least 30% by weight phosphatidylcholine, a polyethylene glycolsurfactant form, additional ethanol, and acacia gum in water to form anadaptogenic herb liposome mixture; combining the third emulsion with themicellular amphiphilic fat and the adaptogenic herb liposome mixture toform a fourth emulsion; combining a pyrroloquinoline quinone saltdissolved in water and sodium hydroxide with the fourth emulsion to forma fifth emulsion; and homogenizing the fifth emulsion under a pressurefrom 100 to 1000 bar to form an aqueous, intra-oral, nanoemulsion blendincluding phosphatidylcholine micelles.

Other systems, methods, features and advantages of the invention willbe, or will become, apparent to one with skill in the art uponexamination of the following figures and detailed description. It isintended that all such additional systems, methods, features, andadvantages be included within this description, be within the scope ofthe invention, and be protected by the claims that follow. The scope ofthe present invention is defined solely by the appended claims and isnot affected by the statements within this summary.

BRIEF DESCRIPTION OF THE FIGURES

The invention can be better understood with reference to the followingdrawings and description. The components in the figures are notnecessarily to scale and are not intended to accurately representmolecules, emphasis instead being placed upon illustrating theprinciples of the invention.

FIG. 1A and FIG. 1B represent a liposome having a double wall ofphospholipids forming a hydrophilic exterior and capsule interior with ahydrophobic wall interior.

FIG. 2 represents a flattened side view of the double wall (bilayer) ofphospholipids that forms the liposome.

FIG. 3 represents a micelle having a single wall of phospholipids(monolayer) forming a hydrophilic exterior and a hydrophobic interiorlacking the capsule interior of a liposome.

FIG. 4 represents a monolayer surfactant where the oil component isassociated with the hydrophobic tails of the surfactant.

DETAILED DESCRIPTION

An aqueous, intra-oral, nanoemulsion blend is provided that enhancesmitochondrial performance in mammals when orally administered. The blendincludes at least two different monolayer surfactant bound particlecomponents and at least one bilayer water-core liposome particlecomponent. The blend optionally may include a micelle.

The at least two different monolayer surfactant bound particlecomponents are oil-in-water dispersions where the oil component of theparticle is associated with the surfactant system. The oil component ofthe particle includes oil-soluble components combined with anassociating oil that assists in associating the oil-soluble componentsof the nanoemulsion blend with the surfactant system. The associatingoil is selected from the group consisting essentially of medium chaintriglycerides (MCT), citrus oil, and combinations thereof.

For the first monolayer surfactant bound particles of the nanoemulsionblend, the oil component is associated with a phosphatidylcholine (PC)and tocopheryl polyethylene glycol succinate (TPGS) surfactant system.The oil component of the first monolayer surfactant bound particlesincludes a cell-signaling pathway enhancement composition in theassociating oil to assist in associating the cell-signaling pathwayenhancement composition with the surfactant system. The cell-signalingpathway enhancement composition is preferably resveratrol or genistein,more preferably resveratrol.

For the second monolayer surfactant bound particles of the nanoemulsionblend, the oil component also is associated with a PC and TPGSsurfactant system. The oil component of the second monolayer surfactantbound particles includes CoQ10 in the associating oil to assist inassociating an electron transport enhancement composition with thesurfactant system. An oil-soluble tocotrienol also may be associatedwith the particles of either or both monolayer surfactant boundparticles. The monolayer surfactant bound particles of the nanoemulsionblend are held in a continuous phase.

The third component of the nanoemulsion blend is a bilayer liposomeoriginating from adaptogenic herbs or from PC. If the nanoemulsion blendincludes the optional micelle, the micelle is primarily formed from PC.Being water-soluble, PQQ may be carried in the continuous water phase,associated with the hydrophilic heads of the PC, or preferably carriedin the interior capsule of a liposome. While formally “water-soluble”the PQQ has a greater affinity for the polar heads of the PC moleculesthan water, when combined in water. When the PQQ is carried in theinterior capsule of a liposome, the liposome is formed primarily fromPC. As discussed further below, the nanoemulsion blend allows the PQQ,resveratrol, CoQ10, and optional tocotrienol to be transported to thecells substantially simultaneously and in the desired ratios to maximizethe enhancement of mitochondrial performance.

Whether the particles of the nanoemulsion blend are monolayer surfactantbound particles or bilayer water-core liposomes, the particles have anaverage diameter from 10 to 125 nanometers (nm), preferably from 10 to80 nm, and more preferably from 10 to 60 nm. The approximately 125-nmaverage diameter upper limit is important, as particles larger than thiswill not transport through the tissues of the mouth, but instead willenter the stomach and be substantially irreversibly chemically altered,and thus deactivated, by acid and bile salts.

Intra-oral delivery of the liposomes in combination with the monolayersurfactant bound particles enables rapid, and substantially simultaneousintra-oral adsorption of both the water-soluble constituents and of theoil-soluble constituents of the nanoemulsion blend into the bloodstream.Thus, intra-oral delivery of the nanoemulsion blend in combination withthe liposome and monolayer surfactant bound particle structures in thecontinuous phase prevents the extensive metabolism of the supplementconstituents of the nanoemulsion blend observed for conventional,orally-administered supplements.

As the constituents of the nanoemulsion blend are transferredintra-orally to the bloodstream without passing through the gut,substantially enhanced bioavailability is achieved. In fact, theliposomes and accompanying monolayer surfactant bound particles of thenanoemulsion blend can approach IV administration in the rate andconcentrations at which the body transfers the supplement constituentsof the nanoemulsion blend into the bloodstream. As the nanoemulsionblend substantially avoids digestion by the stomach, liver andintestine, the delivered supplement constituents enter the bloodstreamsubstantially unaltered. In addition to the advantages of not requiringvenipuncture for relatively rapid and high bloodstream concentrationbioavailability, especially in comparison to conventional oraladministration techniques, the nanoemulsion blend may maintain alonger-duration increased concentration of the delivered supplementconstituents in the bloodstream than available from an IV injection, andthus a longer duration, high-concentration availability to the livingcells.

In addition to water, the aqueous, intra-oral, nanoemulsion blend mayinclude other components including, but not limited to, glycerin,ethanol, sodium hydroxide (NaOH), and a desired flavoring. These othercomponents are selected to not interfere with the beneficial operationof the mitochondrial performance enhancing components or the physicalstructure of the nanoemulsion blend.

Mitochondrial performance, thus the production efficiency of ATP fromglucose and fatty acids within a mammal may be adversely affected bytoxins and aging. The toxins may damage the membranes forming thephysical structures of the mitochondria or interfere with the electrontransport reactions used by the mitochondria to convert fatty acids,glucose, and similar substrates to ATP.

ATP production efficiency also may be adversely affected by age relateddeclines in mitochondrial performance, which generally relate toweakness or deformation of the membranes making up the physicalstructures forming the mitochondria, or a reduction in the ability ofthe mitochondria to produce the necessary enzymes and other agentsnecessary for electron transport. Ninety-five percent of the humanbody's energy is generated through the aerobic conversion (oxidation) ofglucose and fatty acids to ATP within the mitochondria of the cells.

An enhancement of mitochondrial performance per unit time within thecells of a mammal is believed to provide an increase in the health oftissues and organs, in addition to providing enhanced energy, vigor,infection resistance, and weight loss to the mammal. From a tissue/organperspective, the heart, cardiovascular system, brain, central nervoussystem (CNS), muscular, and adrenal systems are believed the primarybeneficiaries. Much of the health benefits provided by an enhancement incellular mitochondrial performance is believed attributable to thesituation where if enough ATP is not present at a given time, the livingcells are forced to ration the available ATP between living,detoxifying, rebuilding their structure, and defending againstinfection. As cells will primarily chose to live, rationed ATP willresult in a retardation in detoxifying, rebuilding, and defendingagainst infection.

The enhancement of mitochondrial performance within the cells of amammal is believed attainable through one or more of three differentpathways. The first pathway is the stimulation of mitochondrialbiogenesis (enhanced creation rate of new mitochondria). An enhancedcreation rate of new mitochondria is believed to provide an enhancementin mitochondrial performance through an increased longevity of the cellin which the mitochondria reside and an increased rate of energyutilization by the cell—thus an increase in base metabolic rate (BMR).In short, the greater the number of mitochondria in the body, thegreater the body's ability to generate ATP per unit time and the morecalories that will be consumed per unit time at the same level ofmusculature and physical activity. From an organ perspective, it isbelieved that the brain and the heart are the greatest beneficiariesfrom mitochondrial biogenesis.

The second pathway to enhanced mitochondrial performance may arise fromenhancing electron transport within the existing mitochondria, thusaccelerating the formation of ATP. The greater the electron transportper unit time within a mitochondria, the greater the amount of ATP thatwill be available to the living cells, and the less likely destructiverationing will occur.

The third pathway to enhanced mitochondrial performance is believedattainable through the protection of existing mitochondria fromradicals, especially when the radicals arise from toxin and heavy metalcatalyzed radical formation. Protecting the existing mitochondria inthis way is believed to increase the activity and lifespan of theexisting mitochondria within the host cell. ATP per unit time generationis believed enhanced through an increase in existing mitochondrialactivity by reducing radical damage to the membrane structures of theexisting mitochondria. The longer the lifespan of the existingmitochondria within a cell, the greater the total number of mitochondriathat will exist within the cell as new mitochondria are produced throughbiogenesis—thus increasing the body's ability to generate ATP per unittime, as previously discussed.

Enhancing the production of mitochondria within cells (mitochondrialbiogenesis) may be accomplished by switching on the genes that producemitochondria. Peroxisome proliferator-activated receptor-gammacoactivator (PGC1a) is believed to enhance the activity of (trigger) thegenes that produce mitochondria. Thus, delivering more bioavailable andbioactive PGC1a to the cells of the body is believed to turn on thesystems that produce mitochondria.

The combined delivery of PQQ and resveratrol to the cells by thenanoemulsion is attributed with an increase in PGC1a activity. In themitochondria, the combination of the PQQ to enhance gene triggering withthe cell-signaling pathway enhancement provided by the resveratrol arebelieved to provide increased mitochondrial biogenesis in relation toeither component in isolation. The lack of the combination of thedifferent functions provided by the PQQ and the resveratrol beingsubstantially simultaneously delivered to the cells is believed toexplain some of the inconsistent results observed in the literature forthese compounds in isolation. PQQ also may be combined with othercell-signaling pathway enhancers, such as genistein.

However, as PQQ is water-soluble, and resveratrol is oil-soluble, theycannot both be delivered to a cell without a carrier system that cantransport both water and oil-soluble constituents. In addition to thecombination of the hydrophilic PQQ with the hydrophobic cell-signalingpathway enhancer, the ratio of these compounds also is believedimportant to achieving the desired mitochondrial biogenesis enhancement.Preferably, the ratio of PQQ to resveratrol is from 1:1 to 1:2,preferably from 1:1.2 to 1:1.8, and more preferably from 1:1.4 to 1:1.6.All ratios are given in terms of weight. To achieve these desired ratiosat the cellular level, the transport of the PQQ and the resveratrol mustbe controlled from introduction to the body until both compounds reachthe interior of the cells. Otherwise, the body will alter the ratiosinconsistently with each introduction as the constituents pass throughthe gut. Preferably, from 3 to 20 mg of PQQ is included in thenanoemulsion blend; however, the different constituents can be differentweights in view of the provided ratios.

Enhancing electron transport within the mitochondria may be accomplishedby the coenzyme Q10 (CoQ10). Unless modified, CoQ10 is oil-soluble.While the literature discusses at length the antioxidant properties ofthis enzyme, we do not believe that the antioxidant properties of theenzyme is the primary function regarding enhancement of mitochondrialperformance.

Oxidative phosphorylation (OXPHOS) is the metabolic pathway in whichcells use enzymes to oxidize nutrients, thereby releasing energy whichis used to form ATP. During OXPHOS, electrons are transferred fromelectron donors to electron acceptors, such as oxygen, in redoxreactions. These redox reactions release energy, which is used to formATP. In the eukaryote cells of mammals, these redox reactions arecarried out by a linked set of protein complexes within the innermembrane of the cell's mitochondria. The linked sets of proteins arecalled electron transport chains. The energy released by electronsflowing through the electron transport chains is used to transportprotons across the inner mitochondrial membrane, in a process calledelectron transport. This generates potential energy in the form of a pHgradient and an electrical potential across this inner mitochondrialmembrane. The store of potential energy within the mitochondria is usedby allowing protons to flow back across the inner mitochondrial membraneand down this gradient, through the ATP synthase enzyme. ATP synthaseconverts the potential energy into ATP. The oxidation of fatty acidswithin the mitochondria may be thought of as mediated combustion, wherethe mitochondria reach temperatures of approximately 50° C. (122° F.).

We believe that CoQ10 operates within the mitochondria to increase therate of electron transport within the interior of the mitochondria. Byincreasing the availability of CoQ10 within the cell and thusavailability to the mitochondria, the rate at which ATP is formed isbelieved to increase. Preferably, the ratio of PQQ to CoQ10 reaching thecells is from 1:2 to 1:4, preferably from 1:2.4 to 1:3.6, and morepreferably from 1:2.8 to 1:3.2. To achieve these desired ratios at thecellular level, the transport of the PQQ and the CoQ10 must becontrolled from introduction to the body until both compounds reach theinterior of the cells. Without the nanoemulsion blend, the body willalter the ratio of PQQ to CoQ10 inconsistently with each introduction asthe constituents pass through the gut.

Protecting the existing mitochondria from oxygen radical damage may beaccomplished with a tocotrienol isomer and with the combination of PQQand the tocotrienol isomer delivered to the cells by the nanoemulsionblend. While the CoQ10 may have some radical inactivation function, aspreviously discussed, the tocotrienol and the combination of PQQ withthe tocotrienol are believed the primary providers of radical protectionprovided to the mitochondria by the nanoemulsion blend.

Unlike the tocopherol forms of Vitamin E, the tocotrienols are believedto demonstrate superior anticancer, immunomodulatory, andneuroprotective properties. The tocotrienol isomers also are believed totarget apoptotic regulators, enzymes, and transcription and growthfactors more effectively. While some beneficial effect may be possibleregarding mitochondria protection with the tocopherol forms of VitaminE, we believe the traits of the tocotrienol isomers to be important tothe enhancement of mitochondrial performance. The delta isomer oftocotrienol is preferred regarding the enhancement of mitochondrialperformance. Presently, we believe it more likely that the tocopherolforms of Vitamin E have a more negative effect on the enhancement ofmitochondrial performance than a benefit.

Although OXPHOS is required for a cell to live, the reaction producesreactive oxygen species, such as superoxide and hydrogen peroxide, whichlead to the formation of potentially membrane-damaging oxygen radicals.If not sufficiently controlled, the oxygen radicals can damage themembrane structures of the mitochondria, thus contributing to theincidence of disease, and possibly aging, as previously discussed.

The membranes and enzymes of the mitochondria have excellent naturalprotection against free radical attack when the free radicals are oxygenbased—thus, the free radicals that naturally occur within themitochondria during ATP production. While this protection could beimproved, it is inherently very good. For example, if CoQ10 is used toincrease electron transport within the mitochondria, an additionalsource of radical deactivation may be helpful to supplement the naturalprotection.

In contrast to oxygen-based free radicals, the membranes and enzymes ofthe mitochondria have poor natural protection to electrophilic toxinsand to other toxin structures that form radicals, as the radicalprotection system inherent to the mitochondria are not conditioned toprotect against electrophilic toxins and toxin-based radicals. Whilemany compounds can form electrophiles in the body, for the mitochondria,electrophilic toxins originating from heavy metals, mold toxins, andendotoxins are of primary consideration. Once electrophilic toxins arepresent within the mitochondria, they can take electrons from the OXPHOSreaction and form radicals within the mitochondria that the natural freeradical protection systems inherent within the mitochondria havedifficulty inactivating. Such electrophilic toxin originated radicalsalso may be longer lived than the oxygen radicals normally occurringwithin the mitochondria due to the molecular structure of theelectrophilic toxin or because the mitochondria do not inherently have asystem to inactivate the electrophilic toxin radicals. Mold basedelectrophilic toxin radicals are also believed to damage themitochondria's inherent oxygen radical defense system, and thus increasethe damage caused by the normally formed oxygen radicals.

The tocotrienol isomers of Vitamin E are believed to more readilyinactivate electrophilic toxin based free radicals than the tocopherolforms of Vitamin E. Thus, in addition to inactivating oxygen andelectrophilic toxin-based radicals, the tocotrienols are believed tobetter protect the inherent oxygen radical protection system of themitochondria and preserve its function against oxygen radicals.Preferred tocotrienols for the enhancement of mitochondrial performanceare the gamma and delta tocotrienols. The ratio of PQQ to tocotrienol isfrom 1:0.1 to 1:2, preferably from 1:0.2 to 1:1, and more preferablyfrom 1:0.3 to 1:0.8. To achieve these desired ratios at the cellularlevel, the transport of the PQQ and the tocotrienol are controlled fromintroduction to the body until both compounds reach the interior of thecells. Without the nanoemulsion blend, the body will alter the ratio ofPQQ to tocotrienol inconsistently with each introduction as theconstituents pass through the gut.

In addition to the described direct action of the tocotrienols inrelation to toxin-based radicals, the PQQ and the combination of thewater-soluble PQQ with the water-soluble tocotrienol is believed tostimulate Nrf2, a nuclear transcription factor. Nrf2 can activate thegenes that increase the production of antioxidants by the cells and thatincrease the production of radical control enzymes. Thus, Nrf2 canincrease the capacity of the natural oxygen radical protection systeminherent to the mitochondria. The more common tocopherol forms ofVitamin E are believed to inhibit Nrf2.

The nanoemulsion blend preferably also includes water- and oil-solubleadaptogenic herbs. The adaptogenic herbs are provided in water includingliposomes. While not believed to act directly on the mitochondria, theability of these herbs to reduce the cortisol response is believed toprovide an added health benefit and to assist in stabilizing thenanoemulsion blend. The adaptogenic herbs preferably included in thenanoemulsion blend include at least three of the following: Acai;Gynostemma (aerial parts) (Jiaogulan); Lycium (fruit) (Himalayan Goji);Maca (root); American Ginseng (root); Siberian Ginseng (root);Schisandra (fruit); Chinese Licorice (root); Rhodiola (root); Astragalus(root); Reishi (fruiting body); Catuaba (bark); Stinging Nettle (aerialparts); Saw Palmetto (fruit); Guarana (seed); Ashwagandha (root);Tribulus (aerial parts); Epimedium (aerial parts); and Yohimbe (bark).When included in the nanoemulsion blend, the adaptogenic herbs morepreferably at least include Gynostemma, American Ginseng, and Rhodiola.

Other herbs and adaptogenic compounds may be included that arechemically compatible with the PQQ, resveratrol, CoQ10, tocotrienol, andthat are physically compatible with the structure of the nanoemulsionblend. While not required, the ratio of PQQ to adaptogenic herbs inwater may be from 1:25 to 1:55, preferably from 1:30 to 1:50, and morepreferably from 1:35 to 1:45. Thus, these ratios are based on the weightof PQQ in view of the weight of the adaptogenic herbs in water.

Phosphatidylcholine (PC) molecules are a subset of the larger set ofphospholipids and are commonly used to form liposomes in water. Whenplaced in water without other constituents, the PC forms liposomes. Theapplication of sufficient shear forces to the PC liposomes can reducethe bilayer liposome structures to monolayer structures.

PC has a head that is water-soluble and a tail that is much lesswater-soluble in relation to the head. PC is a neutral lipid, butcarries an electric dipole moment of about 10 D between the head and thetail, making the molecule itself polar. While “PC” is used throughoutthis document for convenience, PC may be substituted with or combinedwith other amphiphilic fats. Preferable amphiphilic fats are isolatedfrom lecithin, and include glycerophospholipids, phosphatidylcholine,phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid.

Tocopheryl polyethylene glycol succinate 1000 (TPGS) is generallyconsidered a surfactant having a non-polar, oil-soluble “Vitamin E” tailand a polar, water-soluble polyethylene glycol head. While somebeneficial effect may be possible with regard to mitochondria protectionwith the TPGS directly, the oil-soluble tocotrienol forms more readilyprovide the desired concentration and combination of PQQ/tocotrienolwithin the mitochondria in relation to the other components of thenanoemulsion blend. While “TPGS” is used throughout this document forconvenience, TPGS may be substituted with or combined with otherpolyethylene glycol surfactant forms, including polysorbate 40, 60, or80.

The intra-oral delivery of the resveratrol provided by the PC and TPGSstructures of the first monolayer surfactant-based particles of thenanoemulsion blend prevent the extensive metabolism of the resveratrolobserved for conventional oral administration. A modified water-solubleform of CoQ10 could be substituted for the oil-soluble form and thuscarried with the PQQ, but this is not preferred. As the oil-soluble formof CoQ10 is preferred, the PC and TPGS allows the desired ratio of CoQ10to reach the mitochondria, which as with the resveratrol, is otherwiseimpossible with conventional oral delivery. Preferably, the ratio of PQQto PC is from 1:27 to 1:43, preferably from 1:32 to 1:38, and morepreferably from 1:37 to 1:43. While excess PC may be used, it is notrequired.

From a mitochondrial performance enhancement perspective, the PC mayincrease the health of or replace damaged phospholipids in the membranesforming the physical structures of the mitochondria. As efficientelectron transport depends on the mitochondrial membranes having thecorrect physical structures, the PC is believed to enhance electrontransport by improving the integrity of the mitochondrial membranes. TheTPGS may provide some anti-oxidant or electron transport enhancement.However, the primary purpose of the PC and TPGS is to provide anapproximately 125 nm particle average diameter and less transport systemto the mitochondrial performance enhancing components that providesintra-oral component transfer to the bloodstream.

The following examples illustrate one or more preferred embodiments ofthe invention. Numerous variations may be made to the following examplesthat lie within the scope of the invention.

EXAMPLES Example 1: Constituents of the Aqueous, Intra-Oral,Nanoemulsion Blend

A nanoemulsion blend was prepared having a 5 mL total volume. The blendincluded approximately 10 mg of PQQ, 15 mg of resveratrol and 30 mg ofCoQ10 in associating oil, 5 mg of 70% by weight delta tocotrienol, 442mg of PC, 400 mg of adaptogenic herb extract, approximately 500 to 1000mg ethanol, approximately 500 to 2000 mg glycerin, and minor amounts ofNaOH and flavoring. TPGS is included to provide the desired physicalstructures in the nanoemulsion. In addition to these ingredients, theblend included enough water to provide a total volume of 5 mL.

Example 2: A Method of Making an Aqueous, Intra-Oral, Nanoemulsion BlendIncluding PC Micelles

Resveratrol (approximately 15 mg) was solubilized in associating oil,then combined with PC, TPGS, glycerin, and ethanol in water. The mixturewas then homogenized to form an emulsion.

CoQ10 (approximately 30 mg) was in associating oil, then combined withPC, TPGS, glycerin, and ethanol in water. The mixture was thenhomogenized to form an emulsion.

The resveratrol and CoQ10 emulsions were mixed and tocotrienol(approximately 5 mg) was added to the mixed emulsions and mixed.

PC (approximately 400 mg) was added to glycerin and ethanol in water toform micellular PC.

The adaptogenic herb extract including water, ethanol, and glycerin(approximately 400 mg) is combined with PC, TPGS, ethanol, and acaciagum in water to form an adaptogenic herb liposome mixture.

The micellular PC and adaptogenic herb liposomes mixtures were thenadded to and mixed with the mixed emulsions including the tocotrienol.

PQQ (approximately 10 mg) was dissolved in warm water with NaOH and thenadded to the mixture.

The total volume of the mixture was then increased to approximately 5 mLwith water.

The mixture was then subjected to high pressure homogenization fromapproximately 100 to 1000 bar to form the aqueous, intra-oral,nanoemulsion blend including PC micelles.

To provide a clear and more consistent understanding of thespecification and claims of this application, the following definitionsare provided.

Intra-oral delivery means that at least 50%, preferably 60%, and morepreferably 80% and above of the delivery into the bloodstream thatoccurs upon oral administration of the liquid including the deliverableoccurs by transmucosal absorption through the mouth, throat andesophagus before the liquid reaches the stomach. For particles to beconsidered suitable for intra-oral delivery, the average particlediameter is at most 125 nm and preferably less than 80 nm. For example,particles having an average diameter of 100 would have only anapproximately 40% delivery to the bloodstream intra-orally, whileparticles having an average diameter of 75 nm would have and approximate60% intra-oral delivery to the bloodstream. An 80% or greater intra-oraldelivery to the bloodstream may be achieved with an average particlediameter of approximately 50 nm in 0.5 mL liquid after a mouth-residencytime of 2 minutes.

Solutions lack an identifiable interface between the solubilizedmolecules and the solvent. In solutions, the solubilized molecules arein direct contact with the solvent.

Emulsions are mixtures of two or more liquids that do not solubilize.Thus, one of the liquids carries isolated particles in the form ofdroplets of the other liquid. The particles of one liquid may be said tobe dispersed in the continuous phase of the other liquid. An interface,separation, or boundary layer exists between the two liquids, thusbetween the continuous phase and the particles. Emulsions may bemacroemulsions, pseudo-emulsions, microemulsions, or nanoemulsions. Theprimary difference between the types of emulsions is the size (averagediameter) of the particles dispersed in the continuous phase.Macroemulsions and pseudo-emulsions have average particle diameters from1 to 20 micrometers.

Nanoemulsions have average particle diameters from 10 to 125 nanometers,thus being at least an order of magnitude smaller in average particlediameters than macro- and pseudo-emulsions.

In a continuous water phase, all the water molecules are in directcontact with other water molecules, thus providing a continuouslyhydrogen bonded system.

A stable dispersion may be determined in one of two ways. One way toestablish that a dispersion is stable is when the oil phase particles ina continuous water phase do not change in average diameter by more than+/−20% for a time period of at least 3 months to 3 years, preferably fora time period of at least 6 months to 3 years, and more preferably, fora time period of at least 1 year to 3 years. Another way to establishthat a dispersion is stable is when the oil phase particles in thecontinuous water phase do not separate into a visibly distinct phasewith a visible meniscus for a time period of at least 6 months to 3years, and more preferably, for a time period of at least 1 year to 3years.

Average particle diameter is determined by dynamic light scattering(DLS), sometimes referred to a photon correlation spectroscopy. Thedetermination is made between 20 and 25 degrees Celsius. One example ofan instrument suitable for this determination is a Nicomp 380 ZLSparticle sizer as available from Particle Sizing Systems, Port Richey,Fla. DLS can determine the size of particles in a liquid by measuringthe intensity of light scattered from the particles to a detector overtime. As the particles move due to Brownian motion the light scatteredfrom two or more particles constructively or destructively interferes atthe detector. By calculating the autocorrelation function of the lightintensity and assuming a particle distribution, it is possible todetermine the sizes of particles from 1 nm to 5 um. The instrument isalso capable of measuring the Zeta potential of particles.

PGC1-1alpha (PGC1a) is a member of a family of transcriptioncoactivators that plays a central role in the regulation of cellularenergy metabolism. PGC1a stimulates mitochondrial biogenesis andpromotes the remodeling of muscle tissue to a fiber-type compositionthat is metabolically more oxidative and less glycolytic in nature.PGC1a also participates in the regulation of both carbohydrate and lipidmetabolism. It is believed that disorders of PGC1a, such as insufficientexpression, are an underlying contributor to health disorders includingobesity, diabetes, and cardiomyopathy.

Endotoxins are present inside a bacterial cell and are released when thecell disintegrates. Endotoxins are sometimes responsible for thecharacteristic symptoms of a disease.

Heavy metals include mercury, cadmium, lead, arsenic, nickel, chromium,and antimony.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

While various aspects of the invention are described, it will beapparent to those of ordinary skill in the art that other embodimentsand implementations are possible within the scope of the invention.

1. A method of making an aqueous, intra-oral, nanoemulsion blend forenhancing mitochondrial performance in animals when orally administered,the method comprising: homogenizing a mixture comprising acell-signaling pathway enhancement composition, a first portion ofamphiphilic fat including at least 30% by weight phosphatidylcholine, apolyethylene glycol surfactant form, an associating oil, glycerin,ethanol, and water to form a first emulsion; homogenizing a mixturecomprising coenzyme Q10, a second portion of amphiphilic fat includingat least 30% by weight phosphatidylcholine, a polyethylene glycolsurfactant form, an associating oil, glycerin, ethanol, and water toform a second emulsion; combining the first and second emulsions with atleast one tocotrienol isomer of Vitamin E to form a third emulsion;combining a third portion of amphiphilic fat including at least 30% byweight phosphatidylcholine with glycerin and ethanol in water to formmicellular amphiphilic fat; combining an adaptogenic herb extractincluding oil- and water-soluble herbs, water, ethanol, and glycerinwith a fourth portion of amphiphilic fat including at least 30% byweight phosphatidylcholine, a polyethylene glycol surfactant form,additional ethanol, and acacia gum in water to form an adaptogenic herbliposome mixture; combining the third emulsion with the micellularamphiphilic fat and the adaptogenic herb liposome mixture to form afourth emulsion; combining a pyrroloquinoline quinone salt dissolved inwater and sodium hydroxide with the fourth emulsion to form a fifthemulsion; and homogenizing the fifth emulsion under a pressure from 100to 1000 bar to form an aqueous, intra-oral, nanoemulsion blend includingphosphatidylcholine micelles, where the blend is a shelf stabledispersion.
 2. The method of claim 1, where the adaptogenic herb extractcomprises Gynostemma, American Ginseng, and Rhodiola.
 3. The method ofclaim 1, where the associating oil is selected from the group consistingessentially of medium chain triglycerides, citrus oil, and combinationsthereof.
 4. The method of claim 1, where the polyethylene glycolsurfactant form is selected from the group consisting of tocopherylpolyethylene glycol succinate 1000, polysorbate 40, polysorbate 60,polysorbate 80, and combinations thereof.
 5. The method of claim 1,where the polyethylene glycol surfactant form is tocopheryl polyethyleneglycol succinate
 1000. 6. The method of claim 1, where thepyrroloquinoline quinone salt is pyrroloquinoline quinone disodium salt.7. The method of claim 6, where the ratio in the blend of thepyrroloquinoline quinone disodium salt to the coenzyme Q10 is from 1:2to 1:4.
 8. The method of claim 6, where the ratio of thepyrroloquinoline quinone disodium salt to the coenzyme Q10 is from 1:2.8to 1:3.2.
 9. The method of claim 1, where the cell signaling pathwayenhancement composition is selected from the group consisting ofresveratrol, genistein, and combinations thereof.
 10. The method ofclaim 1, where the cell signaling pathway enhancement compositioncomprises resveratrol.
 11. The method of claim 10, where the ratio inthe blend of the pyrroloquinoline quinone salt to the resveratrol isfrom 1:1 to 1:2.
 12. The method of claim 10, where the ratio in theblend of the pyrroloquinoline quinone salt to the resveratrol is from1:1.4 to 1:1.6.
 13. The method of claim 10, where thephosphatidylcholine micelles in the blend have average particlediameters from 10 nm to 125 nm.
 14. The method of claim 10, where thephosphatidylcholine micelles in the blend have average particlediameters from 10 nm to 60 nm.
 15. The method of claim 1, where theratio in the blend of the pyrroloquinoline quinone salt to totalamphiphilic fat including at least 30% by weight phosphatidylcholine isfrom 1:27 to 1:43.
 16. The method of claim 1, where the ratio in theblend of the pyrroloquinoline quinone salt to total amphiphilic fatincluding at least 30% by weight phosphatidylcholine is from 1:37 to1:43.
 17. The method of claim 1, the blend configured to deliver atleast 60% by transmucosal absorption through the mouth of a totalbloodstream delivered concentration of the cell-signaling pathwayenhancement composition, the coenzyme Q10, and the pyrroloquinolinequinone salt, when 0.5 mL of the blend is intra-orally administered to ahuman for a mouth-residency time of 2 minutes.
 18. The method of claim1, the blend configured to deliver at least 80% by transmucosalabsorption through the mouth of a total bloodstream deliveredconcentration of the cell-signaling pathway enhancement composition, thecoenzyme Q10, and the pyrroloquinoline quinone salt, when 0.5 mL of theblend is intra-orally administered to a human for a mouth-residency timeof 2 minutes.
 19. An aqueous, intra-oral, nanoemulsion blend forenhancing mitochondrial performance in an animal when orallyadministered, the blend comprising: a cell-signaling pathway enhancementcomposition delivery means for delivering the cell-signaling pathwayenhancement composition to the bloodstream of the animal; a coenzyme Q10delivery means for delivering the coenzyme Q10 to the bloodstream of theanimal; and a pyrroloquinoline quinone salt delivery means fordelivering the pyrroloquinoline quinone salt to the bloodstream of theanimal, where the cell-signaling pathway enhancement compositiondelivery means and the coenzyme Q10 delivery means both comprise anassociating oil selected from the group consisting essentially of mediumchain triglycerides, citrus oil, and combinations thereof, where thecell-signaling pathway enhancement composition delivery means, thecoenzyme Q10 delivery means, and the pyrroloquinoline quinone saltdelivery means are particles having average particle diameters from 10nm to 80 nm, and where the blend is a shelf stable dispersion.
 20. Theblend of claim 19 further comprising a tocotrienol isomer of Vitamin Edelivery means for delivering a tocotrienol isomer of Vitamin E to thebloodstream of the animal. 21.-30. (canceled)